Design and optimization of fermentation medium for enhanced bacteriocin production by probiotic bacterium Enterococcus faecium MC13

Statistics-based experimental designs were used to develop a cost-effective medium for enhanced production of viable cells and bacteriocin by probiotic Enterococcus faecium MC13. Carbon, nitrogen, and mineral sources were first screened by one-variable-at-a-time (OVAT) methods. In order to increase yield production, the selected variables were further statistically optimized using response-surface methodology (RSM) with central composite design (CCD). The maximum and minimum levels of the selected variables were determined and a set of 34 experimental runs was performed. The optimum concentrations of the tested variables for production of viable cells (12.24 log CFU mL(-1)) and bacteriocin activity (25,600 AU mL(-1)) were tryptone (10.0 g/L), peptone (6.0 g/L), maltose (3.0 g/L), glucose (9.0 g/L), NaCl (15.0 g/L), sodium citrate (2.5 g/L), sodium acetate (1.0 g/L), and dipotassium PO(4) (0.1 g/L). Threefold increased yield of bacteriocin was achieved in optimized medium compared to the unoptimized counterpart, and this was two times less cost than commercial MRS medium.     

Mangiferin: A xanthone attenuates mercury chloride induced cytotoxicity and genotoxicity in HepG2 cells

Mangiferin (MGN), a dietary C-glucosylxanthone present in Mangifera indica, is known to possess a spectrum of beneficial pharmacological properties. This study demonstrates antigenotoxic potential of MGN against mercuric chloride (HgCl2)-induced genotoxicity in HepG2 cell line. Treatment of HepG2 cells with various concentrations of HgCl2 for 3 h caused a dose-dependent increase in micronuclei frequency and elevation in DNA strand breaks (olive tail moment and tail DNA). Pretreatment with MGN significantly (p < 0.01) inhibited HgCl2 -induced (20 μM for 30 h) DNA damage. An optimal antigenotoxic effect of MGN, both in micronuclei and comet assay, was observed at a concentration of 50 μM. Furthermore, HepG2 cells treated with various concentrations of HgCl2 resulted in a dose-dependent increase in the dichlorofluorescein fluorescence, indicating an increase in the generation of reactive oxygen species (ROS). However, MGN by itself failed to generate ROS at a concentration of 50 μM, whereas it could significantly decrease HgCl2 -induced ROS. Our study clearly demonstrates that MGN pretreatment reduced the HgCl2-induced DNA damage in HepG2 cells, thus demonstrating the genoprotective potential of MGN, which is mediated mainly by the inhibition of oxidative stress.     

Biometry based ageing of nestling Indian Spotted Owlets ( Athene brama brama)

Biometric analysis helps in sex differentiation, understanding development and for studies of avian biology such as foraging ecology, evolutionary ecology, and survivorship. We suggest that biometry can also be a reliable, practical and inexpensive tool to determine the age of nestlings in the field by non-invasive methods. As an example we studied the biometry of wing, culmen, talon, tarsus and body mass of nestling southern Indian Spotted Owlets (Athene brama brama). Based on the growth pattern analysis using logistic growth model, discriminant analysis and CHAID (Chi-squared Automatic Interaction Detection) based decision tree, we show that biometry of nestling Spotted Owlets is an easy, reliable and inexpensive method to determine nestling age and to assess growth rate and relative nutritional status. These biometric parameters also allow us to predict their ability to initiate first flight from the nest site. This method is described here for the first time and we postulate that such charts can be devised for other avian species as well, so as to assist conservation biologists and bird rescuers.     

Tissue-specific analysis of glycogen synthase kinase-3α (GSK-3α) in glucose metabolism: effect of strain variation

Background

Over-activity and elevated expression of glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology of insulin resistance and Type 2 diabetes. Administration of specific GSK-3 inhibitors to diabetic or obese rodent models improves glycaemic control and insulin sensitivity. However, due to the indiscriminatory nature of these inhibitors, the relative contribution of the two isoforms of GSK-3 (GSK-3α and GSK-3β) is not known. Recently, we demonstrated that an out-bred strain of mice (ICR) lacking expression of GSK-3α in all tissues displayed improved insulin sensitivity and enhanced hepatic glucose metabolism. We also found that muscle (but not liver) inactivation of GSK-3β conferred insulin and glucose sensitization in an in-bred strain of mice (C57BL/6).

Methodology/Principal Findings

Here, we have employed tissue-specific deletion of GSK-3α, to examine the relative contribution of two insulin-sensitive tissues, muscle and liver, towards the insulin sensitization phenotype originally observed in the global GSK-3α KO animals. We found that mice in which GSK-3α has been inactivated in either skeletal-muscle or liver displayed no differences in glucose tolerance or insulin sensitivity compared to wild type littermates. Given the strain differences in our original analyses, we examined the insulin and glucose sensitivity of global GSK-3α KO animals bred onto a C57BL/6 background. These animals also revealed no significant differences in glucose metabolism/insulin sensitivity compared to their wild type littermates. Furthermore, deletion of hepatic GSK-3α on the out-bred, ICR background failed to reproduce the insulin sensitivity manifested by the global deletion of this isoform.

Conclusions/Significance

From these data we conclude that the improved insulin sensitivity and hepatic glucose homeostasis phenotype observed upon global inactivation of GSK-3α is strain-specific. We surmise that the insulin-sensitization observed in the out-bred strain of mice lacking GSK-3α is mediated by indirect means that do not require intrinsic function of GSK-3α in skeletal muscle and liver tissues.     

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

High-performance capillary electrophoresis for determining HIV-1 Tat protein in neurons

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders.     

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase

Background

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P₁ receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.

Methodology/Principal Findings

Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P_int, and attenuated S1P_ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P_int and potentiated motility of HPAECs to S1P_ext or serum. S1P_ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P_ext or 4-deoxypyridoxine-dependent endothelial cell motility.

Conclusions/Significance

These results suggest S1P_ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.     

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrP^C, into a plethora of assemblies, PrP^Sc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrP^Sc structures. However, the degree to which intra- and inter-strain PrP^Sc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrP^Sc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrP^Sc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrP^Sc particles. We found that each strain had a continuum of PrP^Sc assemblies, with strong correlation between PrP^Sc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrP^Sc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrP^Sc took place at a hydrodynamic radius (R_h) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrP^Sc subpopulations and demonstrate that while PrP^Sc quaternary structure is a major contributor to PrP^Sc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrP^Sc particles from maintaining proteinase K resistance below an R_h of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.     

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 g/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 g/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.